AIDS and AIDS-related disorders (ARC) are caused by a retrovirus, the human immunodeficiency virus (HIV). Barre-Sinoussi et al., "Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for Acquired Immune Deficiency Syndrome (AIDS), Science, 220:868 (1983); and Gallo et al., "Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) From Patients with AIDS and at Risk for AIDS", Science, 224:500 (1984).
Like most viruses, HIV often elicits the production of neutralizing antibodies. Unlike many other viruses and other infectious agents for which infection leads to protective immunity, however, HIV specific antibodies are insufficient to halt the progression of the disease. Therefore, in the case of HIV, a vaccine that elicits the immunity of natural infection could prove to be ineffective. In fact, vaccines prepared from the HIV protein gp160 appear to provide little immunity to HIV infection although they elicit neutralizing antibodies. The failure to produce an effective anti-HIV vaccine has led to the prediction that an effective vaccine will not be available until the end of the 1990's.
The HIV genome has been well characterized. Its approximately 10Kb encodes sequences that contain regulatory segments for HIV replication as well as the gag, pol and env genes coding for the core proteins, the reverse transcriptase-protease-endonuclease, and the internal and external envelope glycoproteins respectively.
The HIV env gene encodes the intracellular glycoprotein, gp160, which is normally processed by proteolytic cleavage to form gp120, the external viral glycoprotein, and gp41, the viral transmembrane glycoprotein. The gp120 protein remains associated with HIV virions by virtue of noncovalent interactions with gp41. These noncovalent interactions are weak, consequently most of the gp120 is released from cells and virions in a soluble form.
Previous studies have shown that the proteins encoded by the gag and especially the env regions of the HIV-1 genome are immunogenic since antibodies to the products of the gag and env genes are found in the sera of HIV infected, AIDS and ARC patients.
It has previously been shown that some antibodies obtained from sera of AIDS and ARC patients, as well as asymptomatic individuals infected with the virus are specific to gp120 and gp160. Occasionally these antibodies are neutralizing. The envelope glycoproteins are the HIV-1 antigen most consistently recognized by antibodies in AIDS and ARC patient sera. Allan et al., "Major Glycoprotein Antigens that Induce Antibodies in AIDS Patients are Encoded by HTLV-III", Science, 228:1091-1094 (1985); and Barin et al., "Virus Envelope Protein of HTLV-III Represents Major Target Antigen for Antibodies in AIDS Patients", Science, 228:1094-1096 (1985). In addition, antibodies in patient sera also recognize epitopes of the viral core proteins encoded by the gag gene.
Immunologically important HIV-1 antigens for use in diagnosis and as potential vaccine compositions have been prepared by cloning portions of the HIV-1 genome in various expression systems such as bacteria, yeast or vaccinia. Cabradilla et al., "Serodiagnosis of Antibodies to the Human AIDS Retrovirus With a Bacterially Synthesized env Polypeptide", Biotechnology, 4:128-133 (1986); and Chang et al., "Detection of Antibodies to Human T-Cell Lymphotropic Virus-III (HTLV-III) With an Immunoassay Employing a Recombinant Escherichia coli - Derived Viral Antigenic Peptide", Biotechnology, 3:905-909 (1985). HIV-1 antigens produced by recombinant DNA methods, however, must still be exhaustively purified to avoid adverse reactions upon vaccination and false positive reactions in ELISA assays due to any antibody reactivity to antigens of the expression system which may contaminate the HIV-1 antigen preparation. Also, denaturation of HIV-1 antigens during purification may destroy important antigen activity. Preparation of proteins from intact viruses can also result in contamination by the virus.
Several publications have presented data showing immunologic reactivity of selected synthetic peptides corresponding to portions of the antigenic proteins of HIV-1. In one study, a peptide having the amino acid sequence Tyr-Asp-Arg-Pro-Glu-Gly-Ile-Glu-Glu-Glu-Gly-Gly-Glu-Arg-Asp-Arg -Asp-Arg-Ser-Gly-Cys which corresponds to amino acid residues 735-752 of HIV-1 was synthesized. Kennedy et al., "Antiserum to a Synthetic Peptide Recognizes the HTLV-III Envelope Glycoprotein", Science, 231:1556-1559 (1986). This peptide, derived from a portion of gp41, was used to immunize rabbits in an attempt to elicit a neutralizing antibody response to HIV-1. Furthermore, several sera from AIDS patients known to contain anti-gp41 antibodies were weakly reactive with this peptide, thus indicating that this peptide contains at least one epitope recognized, to some extent, by antibodies to native gp160/gp41. However, this peptide has not been shown to elicit neutralizing antibodies in mammals other than rabbits nor has it been suggested for use as a human vaccine.
Longitudinal studies conducted on cohorts of HIV-infected individuals have indicated that a stable clinical condition is associated with presence of high titers of neutralizing antibodies against the envelope glycoprotein gp120 of HIV and especially against a specific segment of eight amino acids. Ranki et al., "Neutralizing Antibodies in HIV (HTLV-III) Infection: Correlation with Clinical Outcome and Antibody Response Against Different Viral Proteins", Clin. Exp. Immunol., 69:231 (1987); and Marx (1989).
Achieving protective immunity against HIV is likely to lie on the induction of gp120 specific neutralizing antibodies. Marx, "New Hope on the AIDS Vaccine Front", Science, 244:1254 (1989). Potent T cell help might also be critical to promote the generation and the expansion of virus-specific cytotoxic T cells. Reinherz and Schlossman, "The Characterization and Function of Human Immunoregulatory T Lymphocyte Subsets", Immunol. Today, 2:69 (1981); Burns et al, "Thymus Dependence of Viral Antigens", Nature, 256:654 (1975); and Askonas et al., "Cytotoxic T-memory Cells in Virus Infection and the Specificity of Helper T Cells", Immunology, 45:79 (1982). To be durable and broad, protective immunity should rely on induction of immunologic memory to structurally conserved antigenic moieties comprising epitopes displaying limited MHC restriction for T helper cell recognition. Askonas et al. (1982).
Since production of antibodies, including neutralizing antibodies, by B cells is critically dependent on cognate T cell help, and antigenic determinants recognized by T cells are often distinct from the ones recognized by B cells, identification of antigenic moieties recognized by T cells (so-called "T cell epitopes"), is important when considering vaccination strategies based on appropriate combinations of T and B cell epitopes.
It would therefore be useful in the treatment and prevention of AIDS and ARC to have an HIV vaccine capable of producing neutralizing antibodies and concomitantly eliciting T cell help.
Most antigenic determinants recognized by T cells are composed of continuous stretches of peptides. Berkower et al., "Antigen Conformation Determines Processing Requirements for T-cell Activation", Proc. Natl. Acad. Sci. U.S.A., 79:4723 (1982); DeLisi and Berzofsky, "T Cell Antigen Sites Tend to be Amphipathic Structures", Proc. Natl. Acad. Sci. U.S.A., 82:7048 (1985); and Margalit et al., "Prediction of Immunodominant Helper T Cell Antigenic Sites From Primary Sequence", J. Immunol., 138:2213 (1987). B and T cell recognition sites are often located in different regions of a complex antigen. Milich et al., "Nonoverlapping T and B Cell Determinants on an Hepatitis B Antigert pre-S(2) Region Synthetic Peptide", J. Exp. Med., 164:532 (1986). Within the functional T cell repertoire, T helper cells, T cytotoxic cells, and T suppressor cells, appear to recognize structurally distinct determinants. Krzych et al., "Induction of Helper and Suppressor T Cells by Nonoverlapping Determinants on the Large Protein Antigen, .beta.-galactosidase", FASEB J., 2:141 (1988). This functional separation may have important bearing on the development of vaccines, since particular determinants recognized by T suppressor cells may be ablated resulting in important benefits for immunogenicity.
AIDS and ARC are associated with progressive impairment of CD4.sup.+ T cells, and increased susceptibility to opportunistic infections. In this respect, HIV-infected persons show decreased T helper cell activity for polyclonal B cell differentiation and decreased T cell proliferative responses to antigens and mitogens associated with an early loss of CD29.sup.+ memory T cells. Terpstra et al., "Longitudinal Study of Leukocyte Functions in Homosexual Men Seroconverted for HIV: Rapid and Persistent Loss of B Cell Function After HIV Infection", Eur. J. Immunol., 19:667 (1989); Fahey et al., "Quantitative Changes in T Helper or Suppressor/Cytotoxic Lymphocyte Subsets that Distinguish Acquired Immune Deficiency Syndrome From Other Immune Subsets Disorders", JAMA, 76:95 (1984); Shearer et al., "Functional T Lymphocyte Immune Deficiency in a Population of Homosexual Men Who do not Exhibit Symptoms of Acquired Immune Deficiency Syndrome", J. Clin. Invest., 74:496-506 (1984); Giorgi et al., "Early Effects of HIV on CD4 Lymphocytes in vivo", J. Immunol., 138:3725 (1987); and van Noesel et al., "Functional and Phenotypic Evidence for a Selective Loss of Memory T Cells in Asymptomatic Human Immunodeficiency Virus-infected Men", 86:293 (1990).
The use of synthetic peptides as artificial T cell recognition sites in the composition of candidate subunit vaccines, offers attractive prospects. In this regard, the possibility to educate T helper cells with synthetic peptides for the development of subsequent antibody responses against overlapping and non-overlapping B cell (antibody) recognition sites has been documented in several experimental systems. Berkower et al. (1982); DeLisi and Berzovsky (1985); and Milich et al., "A Single 10-residue PreS(1) Peptide Can Prime T Cell Help for Antibody Production to Multiple Epitopes Within the pre-S(1), pre-S(2), and S regions of HBsAg", J. Immunol., 138:4457 (1987). It has now been found that peptides derived from two regions of the HIV genome elicit T cell activation. These peptides are also capable of inducing the production of neutralizing antibodies to HIV-1.